Human parainfluenza viruses (HPIV), HPIV1, HPIV2, and HPIV3 are significant causes of bronchiolitis, croup and pneumonia in infants and children. Karron et al., J. Infect. Dis. 172: 1445–50 (1995); Collins et al. “Parainfluenza Viruses”, p. 1205–1243. In B. N. Fields et al., eds., Fields Virology, 3rd ed, vol. 1. Lippincott-Raven Publ., Philadelphia (1996); Murphy et al., Virus Res. 11:1–15 (1988). Infections by these viruses result in substantial morbidity in children less than 3 years of age, and are responsible for approximately 20% of hospitalizations among young infants and children for respiratory tract infections.
Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV strain, nor for ameliorating HPIV related illnesses. To date, only two live attenuated PIV vaccine candidates have received particular attention. One of these candidates is a bovine PIV (BPIV) strain that is antigenically related to HPIV3, and which has been shown to protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in human infants and children (Karron et al., J. Inf. Dis. 171:1107–14 (1995a); Karron et al., J. Inf. Dis. 172:1445–1450, (1995b)). A second PIV3 vaccine candidate, JS cp45 is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (Karron et al., (1995b), supra; Belshe et al., J. Med. Virol. 10:235–42 (1982)). This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold-adaptation (ca), and attenuation (att) phenotypes which are stable after viral replication in vivo. The cp45 virus is protective against human PIV3 challenge in experimental animals and is attenuated, genetically stable, and immunogenic in seronegative human infants and children (Hall et al., Virus Res. 22:173–184 (1992); Karron et al., (1995b), supra.
HPIV3 is a member of the Paramyxovirus genus of the Paramyxovirus family, order Mononegavirales. Its genome is a single strand of negative-sense RNA 15462 nucleotides (nt) in length (Galinski et al., Virology 165: 499–510, (1988); Stokes et al., Virus Res. 25:91–103 (1992)) and encodes at least eight proteins (Collins et al., supra, (1996); Galinski, supra, (1991); Spriggs and Collins, J. Gen. Virol. 67: 2705–2719, (1986)). Three of these proteins are associated with the RNA genome to form the nucleocapsid; namely the nucleocapsid protein N, phosphoprotein P, and large polymerase subunit L. Three additional proteins are associated with the envelope, namely the matrix protein M, taught to mediate viral attachment and release, the hemagglutinin-neuraminidase protein HN, and the fusion protein F. Two other proteins, HN and F, represent the neutralizing and protective antigens of PIVs (Collins et al. In Fields Virology, 3rd ed., 1:1205–43 (1996)). Significant sequence divergence in these two protective antigens among different PIVs is the basis for the type specificity of protective immunity against these pathogens (id.).
Another protein of PIV, the C protein, is encoded by an overlapping open reading frame (ORF) of the P protein mRNA (Spriggs and Collins, 1986), and the D protein is generated by RNA editing of the P cistron (Galinski et al. Virology 186:543–50 (1992)). The P mRNA also contains an internal ORF which has the potential to encode a cystein-rich domain called V. The V ORF is also found in other paramyxoviruses and typically is accessed by RNA editing, but this is not the case with PIV. Presently, it is not known whether the PIV V ORF is expressed.
The viral genome of PIV also contains extragenic leader and trailer regions, possessing promoters required for viral replication and transcription. Thus, the PIV genetic map is represented as 3′ leader-N-P/C/D-M-F-HN-L-trailer. Transcription initiates at the 3′ end and proceeds by a sequential stop-start mechanism that is guided by short conserved motifs found at the gene boundaries. The upstream end of each gene contains a gene-start (GS) signal, which directs initiation of its respective mRNA. The downstream terminus of each gene contains a gene-end (GE) motif which directs polyadenylation and termination.
Identification of attenuating mutations in cp45 and other PIV3 vaccine candidates is of interest for a variety of reasons. In particular, it will be useful to understand the genetic basis of attenuation and to characterize the molecular virology and pathogenesis of attenuated HPIV3 strains to provide clinically acceptable vaccines for use against these and other paramyxoviruses, especially HPIV1 and HPIV2 which together account for an additional 7% of pediatric hospital admissions. To achieve these and related goals, a method for producing virus with a wt phenotype from cDNA is needed to determine which mutation(s) in the cp45 virus specify the ts, ca and att phenotypes and which gene(s) of the BPIV3 specify the attenuation phenotype.
The complete nucleotide sequences of the prototype PIV3 strain, and of the JS wt HPIV3 and cp45 strains have been determined (Stokes et al., supra., (1992); Stokes et al., Virus Res. 30: 43–52 (1993)). From these studies, the cp45 strain was shown to possess at least seventeen nucleotide substitutions compared to the parental JS wt HPIV3 strain, eight of which are correlated with changes to viral proteins. However, it has not been previously shown which of these identified mutations specify desired, e.g., ts, ca, and att, phenotypes. Recently, growth of cp45 at nonpermissive temperatures was reported to be complemented by coexpression of a cDNA clone of the L gene of the 47885 wt PIV3 strain (Ray et al., J. Virol. 70:580–584 (1996)), suggesting that the L gene may contain one or more mutations which contribute to the ts phenotype of cp45. However, the results of this study are complicated by the fact that the 47885 strain is not isogenic with the JS parent of cp45 (for example, the two viruses are 4% different at the nucleotide level, and the L proteins differ at 41 amino acid positions (Stokes et al., supra, (1992); published erratum appears in Virus Res. 27:96 (1993); Virus Res. 25:91–103. Also, this method of complementation does not provide a clear measurement of the relative contribution of the L gene mutation(s) to the overall ts phenotype of cp45.
Rescue and analysis of attenuating mutations in PIV3 and other RNA viruses require effective methods to manipulate cDNAs for the particular viruses of interest. Despite previous advancements identifying cDNAs for PIV, manipulation of the genomic RNA of this and other negative-sense RNA viruses has proven difficult. One major obstacle in this regard is that the naked genomic RNA of these viruses is noninfectious.
Successful methods for direct genetic manipulation of non-segmented negative strand RNA viruses have only recently begun to be developed (for reviews, see Conzelmann, J. Gen. Virol. 77:381–89 (1996); Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354–58, (1996)). Functional nucleocapsids have been successfully generated from the intracellular coexpression of separately transfected plasmids bearing the T7 RNA polymerase promoter and encoding either genomic or antigenomic RNA and the N, P, and L proteins. The intracellular cDNA expression is driven by T7 RNA polymerase which is produced by co-infecting with a vaccinia recombinant virus. This approach was first used to determine the transcription and replication requirements of cDNA-encoded minireplicons. Some success has been achieved in the application of these general methods to rescue infectious rabies virus, vesicular stomatitis virus (VSV), measles virus, and Sendai virus from cDNA-encoded antigenomic RNA in the presence of the nucleocapsid N, phosphoprotein P, and large polymerase subunit L (Garcin et al., EMBO J. 14:6087–6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477–81 (1995); Radecke et al., EMBO J. 14:5773–5784 (1995); Schnell et al., EMBO J. 13:4195–203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388–92 (1995)). Respiratory synctial virus (RSV) has also been recovered from cDNA encoded antigenome but this required the transfection of an additional plasmid encoding the M2 ORF 1 transcription elongation factor (Collins et al., 1995).
Rescue of infectious PIV virus and other Mononegavirales members is complicated by virtue of their non-segmented negative-strand RNA genome. The genomic ribonucleoprotein complexes (RNPs) of segmented genome viruses, such as influenza, are generally small in size and loosely structured, and can be assembled in vitro from RNA and required viral proteins. However, PIV and other Mononegavirales members feature much larger and more tightly structured RNPs, which tend to be refractory to functional association in vitro. Furthermore, the coding potential of HPIV3 P mRNA is complicated by cotranscriptional “RNA editing” (Galinski et al., Virology 186: 543–50 (1992)). The resultant shifts in reading frame can access internal ORFs which are expressed as chimeras fused to the N-terminal part of P. In addition, HPIV3 appears to differ from most other paramyxoviruses which express a chimeric V protein, as noted above. The corresponding set of proteins from HPIV3 editing has not yet been identified, and the internal V ORF of HPIV3 is separated from the editing site by numerous translational stop codons (Galinski et al. (1992, supra). Yet another complicating factor is that editing by BPIV3 and HPIV3 produces a novel chimeric protein D, in which the upstream half of P is fused to a domain encoded by a second internal ORF (Pelet et al., EMBO J. 10: 443–448 (1991); Galinski et al., supra, (1992)). The D protein does not have a counterpart in other paramyxoviruses.
In view of the foregoing, an urgent need exists in the art for tools and methods to engineer safe and effective vaccines to alleviate the serious health problems attributable to PIV, particularly illnesses among infants and children attributable to HPIV3. Quite surprisingly, the present invention satisfies these and other related needs.